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Publications - Bioorganic Chemistry

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Preprints

Mot, A. C.; Prell, E.; Klecker, M.; Naumann, C.; Faden, F.; Westermann, B.; Dissmeyer, N.; Real-time detection of PRT1-mediated ubiquitination via fluorescently labeled substrate probes bioRxiv (2016) DOI: 10.1101/062067

The N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turn-over in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway were discovered, ubiquitination mechanism and substrate specificity of N-end rule pathway E3 Ubiquitin ligases remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we use a novel tool to molecularly characterize polyubiquitination live, in real-time.We gained mechanistic insights in PRT1 substrate preference and activation by monitoring live ubiquitination by using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in-gel fluorescence scanning as well as in real time by fluorescence polarization.Enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1-mediated ubiquitination were investigated ad hoc in short time and with significantly reduced reagent consumption.We demonstrated for the first time that PRT1 is indeed an E3 ligase, which was hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.
Publications

Fobofou, S. A. T.; Franke, K.; Porzel, A.; Brandt, W.; Wessjohann, L. A.; Tricyclic Acylphloroglucinols from Hypericum lanceolatum and Regioselective Synthesis of Selancins A and B J. Nat. Prod. 79, 743-753, (2016) DOI: 10.1021/acs.jnatprod.5b00673

The chemical investigation of the chloroform extract of Hypericum lanceolatum guided by 1H NMR, ESIMS, and TLC profiles led to the isolation of 11 new tricyclic acylphloroglucinol derivatives, named selancins A–I (1–9) and hyperselancins A and B (10 and 11), along with the known compound 3-O-geranylemodin (12), which is described for a Hypericum species for the first time. Compounds 8 and 9 are the first examples of natural products with a 6-acyl-2,2-dimethylchroman-4-one core fused with a dimethylpyran unit. The new compounds 1–9 are rare acylphloroglucinol derivatives with two fused dimethylpyran units. Compounds 10 and 11 are derivatives of polycyclic polyprenylated acylphloroglucinols related to hyperforin, the active component of St. John’s wort. Their structures were elucidated by UV, IR, extensive 1D and 2D NMR experiments, HRESIMS, and comparison with the literature data. The absolute configurations of 5, 8, 10, and 11 were determined by comparing experimental and calculated electronic circular dichroism spectra. Compounds 1 and 2 were synthesized regioselectively in two steps. The cytotoxicity of the crude extract (88% growth inhibition at 50 μg/mL) and of compounds 1–6, 8, 9, and 12 (no significant growth inhibition up to a concentration of 10 mM) against colon (HT-29) and prostate (PC-3) cancer cell lines was determined. No anthelmintic activity was observed for the crude extract.
Publications

Fobofou, S. A. T.; Harmon, C. R.; Lonfouo, A. H. N.; Franke, K.; Wright, S. M.; Wessjohann, L. A.; Prenylated phenyl polyketides and acylphloroglucinols from Hypericum peplidifolium Phytochemistry 124, 108-113, (2016) DOI: 10.1016/j.phytochem.2016.02.003

In search for new or chemo-taxonomically relevant bioactive compounds from chemically unexplored Hypericum species, four previously undescribed natural products, named peplidiforones A–D were isolated and characterized from Hypericum peplidifolium A. Rich., together with six known compounds. The structures of all compounds were elucidated by extensive 1D- and 2D-NMR experiments, high resolution mass spectrometric analyses (HR-MS), and by comparison with data reported in the literature. Seven of these compounds are phenyl polyketides while three are acylphloroglucinol type compounds. Peplidiforone C, which possesses an unusual carbon skeleton consisting of a furan ring substituted by a 2,2-dimethylbut-3-enoyl moiety, is the first example of a prenylated furan derivative isolated from the genus Hypericum. The cytotoxicity, antifungal, and anti-herpes simplex virus type 1 (HSV-1) activities of extracts and compounds are described.
Publications

Farag, M. A.; Porzel, A.; Al-Hammady, M. A.; Hegazy, M.-E. F.; Meyer, A.; Mohamed, T. A.; Westphal, H.; Wessjohann, L. A.; Soft Corals Biodiversity in the Egyptian Red Sea: A Comparative MS and NMR Metabolomics Approach of Wild and Aquarium Grown Species J. Proteome Res. 15, 1274-1287, (2016) DOI: 10.1021/acs.jproteome.6b00002

Marine life has developed unique metabolic and physiologic capabilities and advanced symbiotic relationships to survive in the varied and complex marine ecosystems. Herein, metabolite composition of the soft coral genus Sarcophyton was profiled with respect to its species and different habitats along the coastal Egyptian Red Sea via 1H NMR and ultra performance liquid chromatography-mass spectrometry (UPLC–MS) large-scale metabolomics analyses. The current study extends the application of comparative secondary metabolite profiling from plants to corals revealing for metabolite compositional differences among its species via a comparative MS and NMR approach. This was applied for the first time to investigate the metabolism of 16 Sarcophyton species in the context of their genetic diversity or growth habitat. Under optimized conditions, we were able to simultaneously identify 120 metabolites including 65 diterpenes, 8 sesquiterpenes, 18 sterols, and 15 oxylipids. Principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS) were used to define both similarities and differences among samples. For a compound based classification of coral species, UPLC–MS was found to be more effective than NMR. The main differentiations emanate from cembranoids and oxylipids. The specific metabolites that contribute to discrimination between soft corals of S. ehrenbergi from the three different growing habitats also belonged to cembrane type diterpenes, with aquarium S. ehrenbergi corals being less enriched in cembranoids compared to sea corals. PCA using either NMR or UPLC–MS data sets was found equally effective in predicting the species origin of unknown Sarcophyton. Cyclopropane containing sterols observed in abundance in corals may act as cellular membrane protectant against the action of coral toxins, that is, cembranoids.
Publications

Farag, M. A.; Handoussa, H.; Fekry, M. I.; Wessjohann, L. A.; Metabolite profiling in 18 Saudi date palm fruit cultivars and their antioxidant potential via UPLC-qTOF-MS and multivariate data analyses Food Funct. 7, 1077-1086, (2016) DOI: 10.1039/c5fo01570g

Date palm fruit (Phoenix dactylifera) is not only one of the most economically significant plants in the Middle East, but also valued for its nutritional impact, and for which development of analytical methods is ongoing to help distinguish its many cultivars. This study attempts to characterize the primary and secondary metabolite profiles of 18 date cultivars from Saudi Arabia. A total of 44 metabolites extracted from the fruit peel were evaluated in a UPLC-qTOF-MS based metabolomics analysis including flavonoids, phenolic acids and fatty acids. The predominant flavones were glycosides of luteolin and chrysoeriol, as well as quercetin conjugates, whereas caffeoyl shikimic acid was the main hydroxycinnamic acid conjugate. GC-MS was further utilized to identify the primary metabolites in fruits (i.e. sugars) with glucose and fructose accounting for up to 95% of TIC among most cultivars. PCA and OPLS analyses revealed that flavone versus flavonol distribution in fruit were the main contributors for cultivar segregation. The antioxidant activity of date fruit samples was correlated with their total phenolics as determined by DPPH and CUPRAC assays. Dkheni Saudi and Shalabi Madina cultivars, appearing as the most distant in clustering analyses exhibited the strongest antioxidant effect suggesting that multivariate data analysis could help determine which date cultivars ought to be prioritized for future agricultural development.
Publications

Farag, M. A.; Otify, A.; Porzel, A.; Michel, C. G.; Elsayed, A.; Wessjohann, L. A.; Comparative metabolite profiling and fingerprinting of genus Passiflora leaves using a multiplex approach of UPLC-MS and NMR analyzed by chemometric tools Anal. Bioanal. Chem. 408, 3125-3143, (2016) DOI: 10.1007/s00216-016-9376-4

Passiflora incarnata as well as some other Passiflora species are reported to possess anxiolytic and sedative activity and to treat various CNS disorders. The medicinal use of only a few Passiflora species has been scientifically verified. There are over 400 species in the Passiflora genus worldwide, most of which have been little characterized in terms of phytochemical or pharmacological properties. Herein, large-scale multi-targeted metabolic profiling and fingerprinting techniques were utilized to help gain a broader insight into Passiflora species leaves’ chemical composition. Nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) spectra of extracted components derived from 17 Passiflora accessions and from different geographical origins were analyzed using multivariate data analyses. A total of 78 metabolites were tentatively identified, that is, 20 C-flavonoids, 8 O-flavonoids, 21 C, O-flavonoids, 2 cyanogenic glycosides, and 23 fatty acid conjugates, of which several flavonoid conjugates are for the first time to be reported in Passiflora spp. To the best of our knowledge, this study provides the most complete map for secondary metabolite distribution within that genus. Major signals in 1H-NMR and MS spectra contributing to species discrimination were assigned to those of C-flavonoids including isovitexin-2″-O-xyloside, luteolin-C-deoxyhexoside-O-hexoside, schaftoside, isovitexin, and isoorientin. P. incarnata was found most enriched in C-flavonoids, justifying its use as an official drug within that genus. Compared to NMR, LC-MS was found more effective in sample classification based on genetic and/ or geographical origin as revealed from derived multivariate data analyses. Novel insight on metabolite candidates to mediate for Passiflora CNS sedative effects is also presented.
Publications

Faden, F.; Ramezani, T.; Mielke, S.; Almudi, I.; Nairz, K.; Froehlich, M. S.; Höckendorff, J.; Brandt, W.; Hoehenwarter, W.; Dohmen, R. J.; Schnittger, A.; Dissmeyer, N.; Phenotypes on demand via switchable target protein degradation in multicellular organisms Nat. Commun. 7, 12202, (2016) DOI: 10.1038/ncomms12202

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation.
Publications

Edeler, D.; Kaluđerović, M. R.; Dojčinović, B.; Schmidt, H.; Kaluđerović, G. N.; SBA-15 mesoporous silica particles loaded with cisplatin induce senescence in B16F10 cells RSC Adv. 6, 111031-111040, (2016) DOI: 10.1039/C6RA22596A

The anticancer drug cisplatin (CP) is loaded into SBA-15 mesoporous silica (SBA-15|CP) and its release from the nanomaterial is studied. The CP-loaded SBA-15 is tested against four tumor cell lines: mouse malignant melanoma B16F10, human adenocarcinoma HeLa, colon HT-29 and prostate PC3. Most importantly, the superiority of this novel material in comparison to CP arises from the fact that the CP-grafted nanomaterial SBA-15 (→SBA-15|CP) is enhancing cessation of proliferation along with induction of senescence in B16F10 in approximately 3.5 times lower concentration. The control material loaded with therapeutically inactive K2[PtCl4] (→SBA-15|TC) showed no antitumor activity. To a large extent, SBA-15|CP-induced senescence might present a safe approach in tumor treatment. Such cells can be cleared by immune cells resulting in efficient tumor regression. So far only apoptotic agents are being exploited in clinics, thus an understanding of the chemotherapeutic-induced senescence will allow oncologists to explore this essential tumor suppressor mechanism.
Publications

Domik, D.; Thürmer, A.; Weise, T.; Brandt, W.; Daniel, R.; Piechulla, B.; A Terpene Synthase Is Involved in the Synthesis of the Volatile Organic Compound Sodorifen of Serratia plymuthica 4Rx13 Front. Microbiol. 7, 737, (2016) DOI: 10.3389/fmicb.2016.00737

Bacteria release a plethora of volatile organic compounds, including compounds with extraordinary structures. Sodorifen (IUPAC name: 1,2,4,5,6,7,8-heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene) is a recently identified and unusual volatile hydrocarbon that is emitted by the rhizobacterium Serratia plymuthica 4R×13. Sodorifen comprises a bicyclic ring structure solely consisting of carbon and hydrogen atoms, where every carbon atom of the skeleton is substituted with either a methyl or a methylene group. This unusual feature of sodorifen made a prediction of its biosynthetic origin very difficult and so far its biosynthesis is unknown. To unravel the biosynthetic pathway we performed genome and transcriptome analyses to identify candidate genes. One knockout mutant (SOD_c20750) showed the desired negative sodorifen phenotype. Here it was shown for the first time that this gene is indispensable for the synthesis of sodorifen and strongly supports the hypothesis that sodorifen descends from the terpene metabolism. SOD_c20750 is the first bacterial terpene cyclase isolated from Serratia spp. and Enterobacteriales. Homology modeling revealed a 3D structure, which exhibits a functional role of amino acids for intermediate cation stabilization (W325) and putative proton acception (Y332). Moreover, the size and hydrophobicity of the active site strongly indicates that indeed the enzyme may catalyze the unusual compound sodorifen.
Publications

Dieckow, J.; Brandt, W.; Hattermann, K.; Schob, S.; Schulze, U.; Mentlein, R.; Ackermann, P.; Sel, S.; Paulsen, F. P.; CXCR4 and CXCR7 Mediate TFF3-Induced Cell Migration Independently From the ERK1/2 Signaling Pathway Invest. Ophthalmol. Vis. Sci. 57, 56-65, (2016) DOI: 10.1167/iovs.15-18129

Purpose: Trefoil factor family (TFF) peptides, and in particular TFF3, are characteristic secretory products of mucous epithelia that promote antiapoptosis, epithelial migration, restitution, and wound healing. For a long time, a receptor for TFF3 had not yet been identified. However, the chemokine receptor CXCR4 has been described as a low affinity receptor for TFF2. Additionally, CXCR7, which is able to heterodimerize with CXCR4, has also been discussed as a potential TFF2 receptor. Since there are distinct structural similarities between the three known TFF peptides, this study evaluated whether CXCR4 and CXCR7 may also act as putative TFF3 receptors.Methods: We evaluated the expression of both CXCR4 and CXCR7 in samples of human ocular surface tissues and cell lines, using RT-PCR, immunohistochemistry, and Western blot analysis. Furthermore, we studied possible binding interactions between TFF3 and the receptor proteins in an x-ray structure-based modeling system. Functional studies of TFF3–CXCR4/CXCR7 interaction were accomplished by cell culture–based migration assays, flow cytometry, and evaluation of activation of the mitogen-activated protein (MAP) kinase signaling cascade.Results: We detected both receptors at mRNA and protein level in all analyzed ocular surface tissues, and in lesser amount in ocular surface cell lines. X-ray structure-based modeling revealed CXCR4 and CXCR7 dimers as possible binding partners to TFF3. Cell culture–based assays revealed enhanced cell migration under TFF3 stimulation in a conjunctival epithelial cell line, which was completely suppressed by blocking CXCR4 and/or CXCR7. Flow cytometry showed increased proliferation rates after TFF3 treatment, while blocking both receptors had no effect on this increase. Trefoil factor family 3 also activated the MAP kinase signaling cascade independently from receptor activity.Conclusions: Dimers CXCR4 and CXCR7 are involved in TFF3-dependent activation of cell migration, but not cell proliferation. The ERK1/2 pathway is activated in the process, but not influenced by CXCR4 or CXCR7. These results implicate a dependence of TFF3 activity as to cell migration on the chemokine receptors CXCR4 and CXCR7 at the ocular surface.
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